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Clinical Diagnosis Hyperendemic and holoendemic areas Laboratory resources not needed Fever or history of fever Sensitivity ranges from poor to high Often has poor specificity and predictive values Overlap with other syndromes

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Malaria Blood Smear Prepare smears as soon as possible after collecting venous blood to avoid Changes in parasite morphology Staining characteristics Take care to avoid fixing the thick smear Risk of fixing thick when thin is fixed with methanol if both smears on same slide Let alcohol on finger dry to avoid fixing thick Be careful if drying with heat

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Collection of Blood Smears 5. Touch the drop of blood to the slide from below. 4. Slide must always be grasped by its edges. 2. Puncture at the side of the ball of the finger. 3. Gently squeeze toward the puncture site. 1. The second or third finger is usually selected and cleaned.

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Preparing thick and thin films 1. Touch one drop of blood to a clean slide. 2. Spread the first drop to make a 1 cm circle. 3. Touch a fresh drop of blood to the edge of another slide. 6. Wait for both to dry before fixing and staining. 5. Pull the drop of blood across the first slide in one motion. 4. Carry the drop of blood to the first slide and hold at 45  degree angle.

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Estimating Parasite Density Alternate Method Count the number of asexual parasites per high-power field (HPF) on a thick blood film parasites per 100 HPF parasites per 100 HPF parasites per each HPF ++++> 10 parasites per each HPF

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Malaria Antigen Detection - RDTs FeaturePfHRP-2 testspLDH tests Test principle Use of monoclonal (Ab) Detects a histidine rich protein of P.f. Water-soluble protein is released from parasitized RBCs Not present in mature gametocytes Use of monoclonal and polyclonal Ab Detects a parasite enzyme, lactate dehydrogenase pLDH is found in sexual and asexual forms Differentiation between malarial species is based on antigenic differences between pLDH isoforms

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Malaria Antigen Detection - RDTs FeaturePfHRP-2 testspLDH tests AdvantagesThreshold for parasite detection as low as 10 parasites/µl (but sensitivity drops at < 100 parasites /µl) Does not cross react with other species – P.v., P.o., P.m. Threshold for parasite detection ≥ 100 parasites/µl Can detect all species which infect humans Can differentiate between P.f. and non-falciparum malaria Does not cross react with human LDH Positive only in viable parasites, potentially useful for monitoring success of treatment

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Polymerase Chain Reaction (PCR) Threshold of detection at CDC –0.1 parasite/µl if whole blood in tube –2 parasites/µl if using filter paper Definitive species-specific diagnosis now possible Can identify mutations – try to correlate to drug resistance Parasitemia not quantifiable May have use in epidemiologic studies Requires specialized equipment, reagents, and training

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Real-Time PCR New technique based on fluorescence Promising because it has potential to quantify parasitemia, decreases contamination, may detect multiple wavelengths in same tube identifying multiple species in one run, saves hands-on time Needs further research and validation for malaria

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Mass Screening for Malaria in Populations for Resettlement Blood smear examinations to detect asymptomatic parasitemia Not useful for predicting individual risks undetectable parasitemias dormant liver phase parasites Can be used to make a decision about the need for mass treatment of the entire group

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Issues in application of diagnostics Roll Back Malaria objective – At least 60% of those suffering from malaria have prompt access to and are able to use correct, affordable and appropriate treatment within 24 hours of the onset of symptoms Cost should not focus on unit cost alone Must put in context of case management –Amount of drugs being inappropriately dispensed –Increasing drug resistance –Increasingly costly, complex, and toxic alternative drugs –Epidemiology of malaria, populations served –Provider and patient acceptability, esp. of negative results

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Issues in application of diagnostics Rapid diagnostic tests have potential to complement conventional microscopy or provide a diagnostic modality when none is available Operational research is needed to evaluate best uses and cost effectiveness Potential uses –Epidemics and emergencies –Inadequate or absent lab services, unskilled staff –Mobile clinics –Low transmission areas; areas with high levels of drug resistance –Epidemiologic surveys, seroprevalence data